The underlying theme of this proposal is to investigate the cellular mechanisms involved in the regulation of cerebral blood flow (CBF) under physiological conditions as well as in an in vitro model of ischemia. Endothelial cells (EC) are involved in the regulation of the vascular tone in resistance vessels in the brain, while smooth muscle cells (VSM) are the final effectors of both endothelium-dependent and independent CBF changes. Although there have been several indications that the electrophysiological properties of BC have powerful effects on vascular tone, little is known on the ion channel endowment in brain microvascular BC and VSM. We will perform a variety of experiments primarily using the patch clamp technique to investigate the electrophysiological properties of brain microvascular cells, under physiological conditions and after manipulations of intra- and extracellular levels of ATP. In addition, we will test the effects of putative vasodilators on isolated and cannulated brain vessels maintained in vitro. Our preliminary results suggest that intracellular and extracellular ATP play an important role in promoting the release of endothelium-derived relaxing factors via calcium-dependent mechanisms. In particular, we demonstrated the existence in brain endothelial cells of an ATP-sensitive potassium channel (K(ATP)), possibly involved in the regulation of CBF. Our specific aims are: 1a): To investigate the effects of decreased intracellular ATP on vessel diameter. 1b) To investigate the effects of intraluminal application of K(ATP) openers on cerebral arterial vessel diameter. 2) To investigate the electrophysiological effects of K(ATP) agonists on BC and VSM cells. 3) To elucidate the possible involvement of nitric oxide (NO) in the generation of endothelium-mediated, K(ATP) agonist-dependent responses. We hypothesize that during metabolic deprivation and ischemia, decreases in intracellular ATP cause the opening of ATP-sensitive channels, contributing to increases in [Ca+2]i. In addition, we hypothesize that ATP-induced ionic currents may increase the intracellular concentration of calcium sufficient to cause release of NO, leading to vasodilatation. The comprehension of the combination of extracellular and intracellular effects of ATP on brain vascular endothelial cells may add to the understanding of the pathophysiology of stroke.